Lanthanide Labeling of a Potent Protease Activated Receptor‑2 Agonist for Time-Resolved Fluorescence Analysis

Protease activated receptor-2 (PAR₂) is one of four G-protein coupled receptors (GPCRs) that can be activated by exogenous or endogenous proteases, which cleave the extracellular amino-terminus to expose a tethered ligand and subsequent G-protein signaling. Alternatively, PAR₂ can be activated by peptide or peptidomimetic ligands derived from the sequence of the natural tethered ligand. Screening of novel ligands that directly bind to PAR₂ to agonize or antagonize the receptor has been hindered by the lack of a sensitive, high-throughput, affinity binding assay. In this report, we describe the synthesis and use of a modified PAR₂ peptidomimetic agonist, 2-furoyl-LIGRLO-(diethylenetriaminepentaacetic acid)-NH₂ (2-f-LIGRLO–dtpa), designed for lanthanide-based time-resolved fluorescence screening. We first demonstrate that 2-f-LIGRLO–dtpa is a potent and specific PAR₂ agonist across a full spectrum of in vitro assays. We then show that 2-f-LIGRLO–dtpa can be utilized in an affinity binding assay to evaluate the ligand–receptor interactions between known high potency peptidomimetic agonists (2-furoyl-LIGRLO-NH₂, 2-f-LIGRLO; 2-aminothiazol-4-yl-LIGRL-NH₂, 2-at-LIGRL; 6-aminonicotinyl-LIGRL-NH₂, 6-an-LIGRL) and PAR₂. A separate N-terminal peptidomimetic modification (3-indoleacetyl-LIGRL-NH₂, 3-ia-LIGRL) that does not activate PAR₂ signaling was used as a negative control. All three peptidomimetic agonists demonstrated sigmoidal competitive binding curves, with the more potent agonists (2-f-LIGRLO and 2-at-LIGRL) displaying increased competition. In contrast, the control peptide (3-ia-LIGRL) displayed limited competition for PAR₂ binding. In summary, we have developed a europium-containing PAR₂ agonist that can be used in a highly sensitive affinity binding assay to screen novel PAR₂ ligands in a high-throughput format. This ligand can serve as a critical tool in the screening and development of PAR₂ ligands

Lanthanide Labeling of a Potent Protease Activated Receptor‑2 Agonist for Time-Resolved Fluorescence Analysis

Protease activated receptor-2 (PAR₂) is one of four G-protein coupled receptors (GPCRs) that can be activated by exogenous or endogenous proteases, which cleave the extracellular amino-terminus to expose a tethered ligand and subsequent G-protein signaling. Alternatively, PAR₂ can be activated by peptide or peptidomimetic ligands derived from the sequence of the natural tethered ligand. Screening of novel ligands that directly bind to PAR₂ to agonize or antagonize the receptor has been hindered by the lack of a sensitive, high-throughput, affinity binding assay. In this report, we describe the synthesis and use of a modified PAR₂ peptidomimetic agonist, 2-furoyl-LIGRLO-(diethylenetriaminepentaacetic acid)-NH₂ (2-f-LIGRLO–dtpa), designed for lanthanide-based time-resolved fluorescence screening. We first demonstrate that 2-f-LIGRLO–dtpa is a potent and specific PAR₂ agonist across a full spectrum of in vitro assays. We then show that 2-f-LIGRLO–dtpa can be utilized in an affinity binding assay to evaluate the ligand–receptor interactions between known high potency peptidomimetic agonists (2-furoyl-LIGRLO-NH₂, 2-f-LIGRLO; 2-aminothiazol-4-yl-LIGRL-NH₂, 2-at-LIGRL; 6-aminonicotinyl-LIGRL-NH₂, 6-an-LIGRL) and PAR₂. A separate N-terminal peptidomimetic modification (3-indoleacetyl-LIGRL-NH₂, 3-ia-LIGRL) that does not activate PAR₂ signaling was used as a negative control. All three peptidomimetic agonists demonstrated sigmoidal competitive binding curves, with the more potent agonists (2-f-LIGRLO and 2-at-LIGRL) displaying increased competition. In contrast, the control peptide (3-ia-LIGRL) displayed limited competition for PAR₂ binding. In summary, we have developed a europium-containing PAR₂ agonist that can be used in a highly sensitive affinity binding assay to screen novel PAR₂ ligands in a high-throughput format. This ligand can serve as a critical tool in the screening and development of PAR₂ ligands

PAR₂ ligand structures and RTCA EC₅₀.

<p>Compound number, name, structure and the <i>in vitro</i> physiological EC₅₀ (RTCA) of each compound described in the manuscript are shown for comparison. #, compound number; Name, compound name; Structure, compound structure; RTCA, xCELLigence™ real time cell analysis; EC₅₀, half maximal effective concentration; 95% CI, 95% confidence interval.</p

<i>In vitro</i> physiological responses of 16HBE14o- cells following addition of STL agonist compounds 11–14.

<p>Each of the top four panels (<b>A–D</b>) represents physiological responses to agonist compounds as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099140#pone-0099140-g002" target="_blank"><b>Figure 2</b></a>. Concentrations for each experiment (at right of plots) show concentration responses that include supramaximal (black dashed lines) and maximal (solid line) responses. Compounds <b>11</b>: 2at-Cha-IGR-<i>PEG</i>₂-<i>Hdc</i>, and <b>12</b>: 5io-Cha-IGR-<i>PEG</i>₂-<i>Hdc</i> both display rapid and full RTCA responses. However, compounds <b>13</b>: 2at-Cha-I-<i>PEG</i>₂-<i>Hdc</i> and <b>14</b>: 5io-Cha-I-<i>PEG</i>₂-<i>Hdc</i> display delayed responses across concentration ranges that fall short of peak Normalized Cell Index typical for a full agonist. (<b>E</b>) Concentration response curves developed from RTCA using the peak response within the 4 hr experiment are shown in the bottom panel. EC₅₀s for each compound are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099140#pone-0099140-g003" target="_blank"><b>Figure 3</b></a>.</p

<i>In vivo</i> assessment of STL agonist compounds 1–5 induced mechanical hypersensitivity.

<p>Compounds were injected into the plantar surface of the hindpaw at 15 pmoles and mechanical sensitivity was measured at 1 and 3 hr following injection. Compounds <b>1</b>: 2at-LIGRL-<i>PEG</i>₃-<i>Hdc</i>, <b>2</b>: 2at-LIGR-<i>PEG</i>₃-<i>Hdc</i>, <b>3</b>: 2at-LIG-<i>PEG</i>₃-<i>Hdc</i> and <b>4</b>: 2at-LI-<i>PEG</i>₃-<i>Hdc</i> evoked mechanical hypersensitivity whereas Compound <b>5</b> (E; 2at-L-<i>PEG</i>₃-<i>Hdc</i>) was inactive. Compound <b>1</b> caused mechanical hypersensitivity in PAR₂ WT mice but was inactive in PAR₂^-/- mice (F). * p<0.05.</p

Ca²⁺ signaling responses for STL agonist compounds 1–4.

<p>The top four panels (<b>A–D</b>) display traces from a single experiment of average individual cell [Ca²⁺]_i (± SEM) over time for 16HBE14o-cells exposed to PAR₂ STL agonist compounds <b>1–4</b>. Concentrations (Compound <b>1</b>: 2at-LIGRL-<i>PEG</i>₃-<i>Hdc</i>, 15 nM; <b>2</b>: 2at-LIGR-<i>PEG</i>₃-<i>Hdc</i>, 15 nM; <b>3</b>: 2at-LIG-<i>PEG</i>₃-<i>Hdc</i>, 200 nM; <b>4</b>: 2at-LI-<i>PEG</i>₃-<i>Hdc</i>, 2 µM) were chosen to reflect minimal agonist concentration necessary to result in 95% activation of 16HBE14o- cells (n≥3 for each compound). The known peptidomimetic full agonist, (<b>E</b>) 2at-LIGRL-NH₂ (10 µM) is shown for comparison. Although all compounds displayed full Ca²⁺ activation over the 5 min experiment (<b>F</b>), compounds <b>3</b> and <b>4</b> displayed a slight delay in average time to peak Ca²⁺ response (<b>G</b>).</p

Synthetic route for 2at-LIGRL-<i>PEG</i>₃-<i>Hdc</i> (compound 1). <i>i</i>

<p>Reductive alkylation: hexadecyl amine (5 equivalents), sodium cyanoborohydride (5 equivalents) in 5% acetic acid in DCM (0.25 M solution), overnight; <b><i>ii</i></b> a) Fmoc-<i>PEG</i>(6 equiv), DIC (3 equivalents) for first coupling b) Piperidine/DMF (1∶9) for Fmoc deprotection <b><i>iii</i></b> Fmoc/tBu synthesis continued as follows: a) Fmoc-aa-OH (3 equivalents) activated by HOBt (3 equivalents), DIC (3 equivalents), or HBTU (3 equivalents), DIEA (6 equivalents) in DMF; b) Piperidine/DMF (1∶9) for Fmoc deprotection; <b><i>iv</i></b> TFA-scavenger cocktail (91%), water (3%), triisopropylsilane (3%), and thioanisole (3%) for 4 hr.</p

Concentration response curves for STL agonist compounds 1–4.

<p>Concentration response curves were developed from i<i>n vitro</i> physiological responses (RTCA) using the peak response within the 4 hr experiment. Compounds <b>1</b> (2at-LIGRL-<i>PEG</i>₃-<i>Hdc</i>) and <b>2</b> (2at-LIGR-<i>PEG</i>₃-<i>Hdc</i>) have roughly equivalent EC₅₀s (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099140#pone-0099140-g003" target="_blank"><b>Figure 3</b></a>), while further truncated compounds <b>3</b> (2at-LIG-<i>PEG</i>₃-<i>Hdc</i>) and <b>4</b> (2at-LI-<i>PEG</i>₃-<i>Hdc</i>) have higher EC₅₀s (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099140#pone-0099140-g003" target="_blank"><b>Figure 3</b></a>).</p

Ca²⁺ desensitization responses for STL agonist compounds 11–14.

<p>The top four panels (<b>A–D</b>) display traces of the average change in [Ca²⁺]_i. for all cells in the field of view plotted over time (10 min). In each panel, PAR₂ desensitization with 50 µM 2at-LIGRL-NH₂ prevented Ca²⁺ signaling by a second application of 2at-LIGRL-NH₂ and subsequent addition of PAR₂ STL agonists — Compound <b>11</b>: 2at-Cha-IGR-<i>PEG</i>₂-<i>Hdc</i>, 200 nM; <b>12</b>: 5io-Cha-LIG-<i>PEG</i>₂-<i>Hdc</i>, 50 nM; <b>13</b>: 2at-Cha-I-<i>PEG</i>₂-<i>Hdc</i>, 600 nM; <b>14</b>: 5io-Cha-I-<i>PEG</i>₂-<i>Hdc</i>, 300 nM were monitored. Subsequent application of 5 µM ATP in each experiment demonstrated that Ca²⁺ response was intact, and only PAR₂ dependent pathways were desensitized. In the bottom four panels (<b>E–H</b>) 16HBE14o- cells were desensitized with the STL compounds <b>1–4</b> at 10 fold their full activation concentrations — Compound <b>11</b>: 2at-Cha-IGR-<i>PEG</i>₂-<i>Hdc</i>, 2 µM; <b>12</b>: 5io-Cha-LIG-<i>PEG</i>₂-<i>Hdc</i>, 500 nM; <b>13</b>: 2at-Cha-I-<i>PEG</i>₂-<i>Hdc</i>, 6 µM; <b>14</b>: 5io-Cha-I-<i>PEG</i>₂-<i>Hdc</i>, 3 µM. Although compounds <b>11</b> and <b>12</b> were effective in desensitizing 16HBE14o- cells to 10 µM 2at-LIGRL-NH₂, desensitization by compounds <b>13</b> and <b>14</b> was incomplete. In each case, responses to 5 µM ATP remained fully intact. (<b>I</b>) Further examination of Ca²⁺ responses in 16HBE14o- cells demonstrated an incomplete Ca²⁺ activation for compounds <b>13</b> and <b>14</b> persisted at the high agonist concentrations used to desensitize the cells. These data support PAR₂ specificity for each compound, however the heterocycle-dipeptide STLs do not support full agonistic responses.</p

Selectivity of parent compound 2at-LIGRL-NH₂ and potent STL agonist compounds 1, 2 for PAR₂ over MrgprC11.

<p>Compounds were applied to PAR₂ deficient CHO cells transfected with MrgprC11 and evaluated for Ca²⁺ response. Compound <b>1</b>: 2at-LIGRL-<i>PEG</i>₃-<i>Hdc</i> was able to induce a full Ca²⁺ response at the highest concentration tested that was negligible concentrations typically used for PAR₂ activation (e.g., 1<b>–</b>10 nM). Both the parent compound (2at-LIGRL-NH₂) and the Leu₆-truncated STL (compound <b>2</b>: 2at-LIGR-<i>PEG</i>₃-<i>Hdc</i>) displayed limited MrgprC11 activity. None of the compounds displayed activity in untransfected CHO cells (not shown). Each column represents three experiments, each with ∼200 cells.</p